Efficiency of enzymes and benzalkonium chloride treatments against Listeria monocytogenes dual-species biofilms determined by fluorescence microscopy and image analysis

In the present study, three different mixed-species Listeria monocytogenes-carrying biofilms previously isolated from surfaces of seafood, meat and dairies industrial environments were selected in order to test the efficiency of cleaning and disinfection procedures based on the combination of an enzymatic solution, followed by a treatment with benzalkonium chloride (BAC). Biofilm samples were cultured onto AISI-316 type stainless steel 1 cm2 coupons in a batch system at 25 degrees and the effect of sequential exposure to different concentrations of Pronase – BAC, Cellulase – BAC or DNAseI – BAC solutions was then assessed in mature biofilms after 168 hours in nine different experiments using an orthogonal first order approach. Treated samples were stained using then LIVE/DEAD Biofilm viability kit (Invitrogen) and the efficiency of each individual treatment was then determined by epifluorescence microscopy and subsequent image analysis of 25-field mosaics representing a total surface of 1.92mm2, so as to quantify the remaining living cells attached to the surface of the coupon. Analysis of data reveals that synergistic effects are observed after using Pronase – BAC on biofilms coming from fish and dairy industries obtaining almost a total cell detachment in the latter case, whereas DNAse – BAC treatments are efficient only against fish industry biofilms. No significant effect was observed among the Cellulase – BAC procedures, being in some cases even counter-productive. In addition to this, individual effects of Cellulase and Pronase are observed on fish and fish and dairy industry biofilms respectively demonstrating its potential as a cleaning agent even at low concentrations of the enzyme. These results show how the usage of customised treatments combining commercially available enzymes with classical chemically-based disinfection procedures may represent an attractive and efficient alternative against bacterial biofilms in food industry.